Hepatic stellate cell precursors and methods of isolating same

ABSTRACT

The present invention relates to precursor cells to hepatic stellate cells, compositions comprising same and methods of isolating same. The surface antigenic profile of the precursors is MHC class Ia negative, ICAM-1+, VCAM-1+, β3-integrin+. In addition to expression of these surface markers, the cells also express the intracellular markers desmin, vimentin, smooth muscle α-actin, nestin, hepatocyte growth factor, stromal derived factor-1α and Hlx homeobox transcriptional factor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/337,092, filed Jul. 21, 2014; which is a continuation of U.S. patentapplication Ser. No. 12/903,824, filed Oct. 13, 2010; which is adivisional application of U.S. patent application Ser. No. 11/753,326,filed May 24, 2007, now U.S. Pat. No. 7,824,911; which claims priorityto US Provisional Application No. 60/808,548, filed May 26, 2006, thedisclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates generally to precursors of cells thatcomprise a mature liver. More particularly, the present inventionrelates to precursor cells to hepatic stellate cells, compositionscomprising same and methods of isolating same.

BACKGROUND OF THE INVENTION

Hepatic stellate cells (HpStCs) were first described by Kupffer in the19th century and were designated as “Sternellen” for their “stellar”sparkle when viewed under a microscope. HpStCs are liver-specificmesenchymal cells found in the Space of Disse and are comprised, insignificant part, of cytoplasmic lipid droplets containing vitamin A. Infact, the lipid droplets contribute to the “sparkle” quality associatedwith HpStCs.

It is now accepted that HpStCs play a major role in the uptake, storageand release of vitamin A compounds, which are necessary particularly forvision, reproduction, and embryonic development. In mammals, about 50 to80% of the total body vitamin A is normally stored in HpStCs.

HpStCs also play a central role in the production of growth factors,extracellular matrix components (ECMs), and matrix metalloproteinases inliver. A number of reports demonstrate that HpStCs secrete severalmitogens for hepatocytes—such as EGF, TGFα and HGF—and play a centralrole in liver development and regeneration. Similarly, a numbers ofstudies demonstrate that an imbalance in ECM regulation is a factor inliver fibrosis or cirrhosis. Furthermore, the contractile properties ofHpStCs suggest that they have a similar function to the pericytes, whichcontrol local blood flow in blood vessels. Taken together, these diversefunctions of HpStCs illustrate their significant role in healthy anddysfunctional hepatic function.

Despite our growing understanding of the importance of HpStCs, theorigin of HpStCs remains unknown. In early liver development, endodermalcells in the foregut give rise to hepatic diverticulum, which, in turn,develops into surrounding mesoderm called the septum transversum andforming the hepatic cords. While some have presumed that HpStCprogenitors could derive from mesenchymal cells in the septumtransversum, no HpStCs have been isolated from it, and surface markersenabling immunoselection and/or characterizing precursor HpStCs have yetto be identified.

Accordingly, there is a need for markers that specifically identifyprecursors to HpStCs and for a method of isolating same with saidmarkers. In addition, there is a need for a method of propagating HpStCprecursor cells in vitro.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, a method of obtaining apopulation of cells enriched in hepatic stellate cell progenitor cellsis provided comprising (a) providing a single cell suspension of cellsfrom mammalian tissue; and sequentially, in any order, or substantiallysimultaneously, (b) removing from the single cell suspension those cellsthat express MHC class Ia antigen; and (c) isolating from the cellsuspension those cells that are positive for Vitamin A fluorescence, toobtain a population of cells enriched in hepatic stellate cellprogenitors. The mammalian tissue may be liver, pancreas, gut, lung, orbone marrow cells, preferably liver. The method may further compriseisolating from the cell suspension those cells that are positive forVCAM and/or β3-integrin; removing from the cell suspension those cellsthat express CD45; and/or isolating from the cell suspension those cellsthat express desmin, nestin, vimentin, smooth muscle alpha-actin or acombination thereof.

In some embodiments, the isolating and removing steps are carried out ina flow cytometer. Removal of cells that express MHC class I antigens maybe carried out with a species-specific antibody against cells expressingthose antigens; for example, utilizing antibodies against RT1A in ratliver cells. As well, the hepatic stellate progenitor cells may be humanhepatic stellate cell progenitors.

In yet another aspect of the present invention, a method of obtaining apopulation of cells enriched in isolated hepatic stellate cellprogenitors is provided comprising (a) obtaining a cell suspension ofhepatic cells; and (b) sequentially, in any order, or substantiallysimultaneously, (i) isolating from the single cell suspension of livercells those cells that are positive for ICAM-1 antigen (ii) removingthose cells that are positive for MHC class I antigen, and (iii)isolating those cells that are positive for Vitamin A fluorescence asmeasured in a flow cytometer, to obtain a population of cells enrichedin progenitors. The method may further comprise removing from the cellsuspension those cells that express MHC class I antigen, CD45 or bothand/or isolating from the cell suspension those cells that expressdesmin, nestin, vementin, smooth muscle alpha actin or a combinationthereof.

In yet another embodiment of the present invention, an isolated hepaticstellate precursor cell which expresses both VCAM antigen andβ3-integrin antigen is provided. In still further yet another embodimentof the present invention, a method of clonogenic expansion of stellateprecursor cells is provided comprising culturing isolated stellateprecursor cells expressing both VCAM antigen and β3-integrin antigen inserum-free media. The media may further comprise a growth factor, suchas, for example, insulin, transferrin, leukemia inhibitory factor (LIF)or epidermal growth factor (EGF) or a combination thereof. The isolatedstellate precursor cells may be further cultured in the presence offeeder cells, for example, STO cells.

In this respect, before explaining at least one embodiment of theinvention in detail, it is to be understood that the invention is notlimited in its application to the details of construction and to thearrangements of the components set forth in the following description orillustrated in the drawings. The invention is capable of embodiments inaddition to those described and of being practiced and carried out invarious ways. Also, it is to be understood that the phraseology andterminology employed herein, as well as the abstract, are for thepurpose of description and should not be regarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that the claims be regarded as including such equivalentconstructions insofar as they do not depart from the spirit and scope ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show flow cytometric analysis for autofluorescent cells in13 dpc rat fetal liver and lung. FIG. 1A shows the pattern of forwardscatter (FSC) and side scatter (SSC) of the entire population (ALL).Based on the value of SSC, R1 and R2 gates were created and representedhigh (SSC^(hi)) and low)(SSC^(lo)) SSC, respectively. Expressionpatterns of RT1A and ICAM-1 in the R1 and R2 are also shown.RT1A⁻ICAM-1⁺ SSC^(hi) cells (R2, lower right) are hepatoblasts in therat fetal liver (Kubota and Reid, 2000). The number indicates percentageof each quadrant. FIG. 1B shows the autofluorescent pattern of entirepopulation (ALL), R1, and R2 were analyzed with UV laser and 488 nmlaser. UV laser specific autofluorescent signal was detected with a 450nm filter, while non-specific autofluorescent signal excited with a 488nm laser was measured with a 530/30 bandpass filter. UV laser-specificautofluorescent cells were detected in R1 and R2 (upper left). FIG. 1Cshows the expression of RT1A and UV laser specific autofluorescentsignal was studied. UV laser-specific autofluorescent cells were RT1A⁻.ns-autoflu⁺RT1A⁻ cells (allow) were identified and were correspond torat hepatoblast population. FIG. 1D shows a UV laser specificautofluorescent signal was analyzed in 13 dpc fetal lung cells. Thereare no UV specific autofluorescent cells in the lung cell population.Most of all cells are RT1A⁻, and no non-specific autofluorescent cells(comparable to the hepatoblast population in the fetal liver) weredetected.

FIGS. 2A and 2B show VCAM-1 and ICAM-1 expression on vA⁺ cells. FIG. 2Ashows a Histogram of flow cytometry for VCAM-1 expression on 13 dpcfetal liver. Approximately 15% of cells express VCAM-1 on the cellsurface. Closed and open histograms represent stained cells andunstained cells, respectively. VCAM-1⁺ and VCAM-1⁻ cells were analyzedby flow cytometry for their autofluoresent signals. All vA⁺ cells andns-autoflu⁺ cells are VCAM-1 positive. The numbers represent thepercentage of each quadrant. FIG. 2B shows the two color analysis of 13dpc fetal liver cells for RT1A and ICAM-1. R1 cell population(RT1A⁻ICAM-1⁺) contains all vA⁺ cells and ns-autoflu⁺ cells. Theseresults indicate that vA⁺ and ns-autoflu⁺ cells are VCAM-1⁺RT1A⁻ICAM-1⁻.

FIGS. 3A and 3B show antigenic profiles of vA⁺, ns-autoflu⁺, andautoflu⁻RT1A⁻ cells in 13 dpc fetal liver. FIG. 3A shows the flowcytometric analysis for UV-autofluorescence and RT1A expression. In theRT1A⁻ cell population, four gates (R1-R4) were created based on theautofluorescent signals. FIG. 3B shows a two color analysis of VCAM-1versus β3-integrin, PECAM-1, or Thy-1 expression for each gated cellpopulation (R1-R4). The numbers represent the percentage of eachquadrant. Primarily R1 cells are VCAM-1⁺ β3-integrin⁺, while R3 cellsuniformly express VCAM-1, but none of β3-integrin, PECAM-1, or Thy-1.

FIG. 4 shows immunocytochemistry of a bipotent hepatblast colony.ns-autoflu⁻ VCAM-1⁺ cells were isolated by FACS and placed on STO feedercells in HDM at a clonal cell density (250 cells in a well of 12-wellplate; 66 cells/cm²). After 15 days in culture, the cells were fixed andstained with antibodies ageainst ALB (red) and CK19 (green). Each colonywas generated from a single sorted cell (Kubota and Reid, 2000). Morethan 95% (95.7±0.4%; mean±SEM, n=3) of hepatic colonies contained ALB⁺CK19⁻ and ALB⁻CK19⁺ cells, which represent hepatocytic and biliarydifferentiation, respectively.

FIG. 5 provides RT-PCR analysis of 14 dpc fetal liver cells fractionatedby FACS. Lane 1, ns-autoflu⁺RT1A⁻VCAM-1⁺β 3-integrin⁻; lane 2,vA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺; lane 3, autoflu⁻RT1A⁻VCAM-1⁺; lane 4,autoflu RT1A⁻VCAM-1⁻; lane 5, remaining VCAM-1⁻ cell population; lane 6,no cDNA. vA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺ cells express SDF-1α and HGFstrongly. The vA⁺ cells are positive for HpStC markers (desmin, nestin,vimentin, SMαA) and negative for hepatoblast markers (albumin andProx1).

FIG. 6 shows the effect of LIF and EGF on in vitro proliferation ofvA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺ cells. (A) Five hundred vA⁺RT1A⁻VCAM-1⁺β3-integrin⁺ cells isolated by FACS were placed in a well of 96-wellplate with HDM plus laminin supplemented LIF and/or EGF at theconcentration indicated. After 5 days-culture, degree of cellproliferation was measured by the tetrazolium salt WST-1. LIF supportproliferation of the vA⁺ cells at as low as 0.1 ng/ml. EGF slightlyimproved the vA⁺ cell proliferation. (B) Two hundred fiftyRT1A⁻VCAM-1⁺β3-integrin⁺vA⁺ cells isolated by FACS were seeded on STOfeeder cells in HDM with EGF and/or LIF. Twelve-well plates were used.The cultures were stained with Diff-Quick™ after 2-week culture period.Although STO cells express LIF, the amount of the production was notadequate to support clonal expansion of the cells in the absence ofexogenous LIF supplementation. Exogenous LIF and addition of EGFdramatically improved clonal expansion of the vA⁺ cells.

FIG. 7 shows the immunocytochemistry of colonies derived fromvA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺ cells isolated by FACS. Cells were placedon STO feeders in HDM supplemented with EGF and LIF. Fifteen days afterin vitro culture, cultures were stained with antibodies for desmin ornestin. Colony forming cells express nestin and desmin, whereas STOcells do not express either.

FIG. 8 shows the immunocytochemistry of 2-month cultured vA⁺RT1A⁻VCAM-1⁺β3-integrin⁺ cells isolated by FACS. Sorted cells were placed on STOfeeders in HDM supplemented with EGF and LIF. Proliferating cells weresubcultued 5 times on fresh STO feeders. Cultured cells were stainedwith antibodies for desmin or nestin. Proliferating cells maintain theexpression of nestin and desmin during the culture period.

FIG. 9 provides phenotypic characteristics of 2-month culturedvA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺ cells. (A) RT-PCR analysis of culturedvA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺ cells. Cells were isolated by FACS andcultured on STO feeders in the HDM with EGF and LIF. After 2-monthculture cells were fractionated by FACS. Proliferating rat cells andmouse STO feeder cells were fractionated by FACS following antibodystaining of mouse CD98 monoclonal antibody. CD98 is expressed on mouseSTO cells, and the monoclonal antibody reacts specifically mouse CD98,but not rat CD98. RNAs were isolated from vA⁺-derived rat cells and STOcells. Normal rat HpStCs were also used and isolated the RNA for acontrol. cDNAs were synthesized from those RNAs and subjected to PCRwith primers specific for various transcripts that expressed in HpStCs.(B) Flow cytometry for cultured vA⁺RT1A⁻VCAM-1⁺ β3-integrin⁻ cells.Cells used for RT-PCR were stained with anti-VCAM-1 or RT1A antibody andmouse CD98 antibody. The CD98 negative fraction was analyzed for VCAM-1or RT1A expression. Continuously proliferating cells derived from vA⁺cells in rat fetal livers express VCAM-1 and RT1A uniformly under theculture condition examined.

DETAILED DESCRIPTION OF THE INVENTION

HpStCs have been assigned various names, including “lipocytes,”“fat-storing cells,” “Ito cells,” “peri-sinusoidal cells,” and “liverpericytes.” In the interest of clarity, however, only the term HpStCwill be used in this paper, which should nonetheless be understood torefer to the same population of cells having any and all of theaforementioned alternate names. As well, the teachings herein are notlimited to any one species. In fact, it should be understood that theexamples provided herein are merely exemplary and should not beconstrued as limiting. The instant invention, in this way, is notlimited by its mammalian source for liver tissue. Mammals from which theHpStCs and their precursors may be derived include, but are not limitedto, humans, rodents (e.g., rats, mice, hamsters), rabbits, bovines,horses, pigs, and sheep. Preferably, the HpStCs and their precursors arederived from humans. Nor is the instant invention limited to anyparticular stage of liver development. Thus, the instant invention maybe practiced with fetal, neonatal, pediatric and/or adult liver tissue,including liver tissue from recently deceased individuals (e.g., lessthan about 30 hours post mortem).

The instant invention provides techniques for the isolation andpropagation of HpStC precursor cells (also referred to herein as “HpStCprecursors” or “precursor HpStCs”). HpStC precursors in rat fetal liverwere identified by flow cytometry using the specific auto-fluorescencegenerated by cytoplasmic vA rich lipid droplets. The surface phenotypeof vA⁺ cells appeared to be uniform, and they were RT1A⁻ICAM-1⁺ VCAM-1⁺β3-integrin⁺ PECAM-1⁻. In addition to those surface markers, vA⁺ cellsexpress intermediate filaments specific for HpStCs including desmin,vimentin, SMαA, and nestin.

Although ICAM-1 expression on fetal liver cells is broad, β3-integrin isrelatively specific on vA⁺ cells. β3-integrin requires α-integrin,αv-integrin or αII-integrin, for the surface expression. The choicevaries with the cell types. In the case of HpStCs in adult liver,αv-integrin is used for the α-chain. Therefore, it is likely that HpStCprecursors express αv-integrin. Interestingly, interaction ofαvβ3-integrin expressed on adult HpStCs and the ECM ligands appeared toinfluence the fate determination of HpStCs, proliferation or apoptosis.The αvβ3-integrin transduced a stimulatory signal to protect apoptoticresponses in adult HpStCs. In addition, another report showed thatαvβ3-integrin binds PECAM-1. Thus, without being limited to or bound bytheory, β3-integrin expression on HpStC precursors seems to be importantto receive stimulatory signals from surrounding ECM ligands orendothelial cells, which express PECAM-1, for proliferation during fetalliver development.

While FACS analysis indicated that high VCAM-1 expression was detectedon hepatoblasts and HpStC precursors in fetal liver, the later may playmore important roles for hematopoietic cells, because they expressSDF-1α as well. SDF-1α is a potent chemoattractant for hematopoieticstem cells, which express CXCR4, the receptor for SDF-1α. The chemokineplays a central role during the migration of hematopoieticstem/progenitor cells to bone marrow and is thought to up-regulate VLA-4dependent adhesion to VCAM-1. Therefore, it is possible that SDF-1α andVCAM-1 expression on HpStC precursors are crucial to recruithematopoietic stem/progenitor cells into fetal liver.

Interestingly, VCAM-1 is expressed on hepatoblasts. In addition to thesurface phenotype and mRNA expression, in vitro CFA for hepatoblastsdemonstrated that VCAM-1⁺ cells are hepatoblasts. This finding isunexpected because VCAM-1 is known as a surface marker for mesenchymalcells such as endothelial cells, myogenic cells, or HpStCs. Theexpression appears to be developmentally controlled because adulthepatocytes are VCAM-1⁻ by FACS analysis.

It appears that HpStC precursors are important for liver development,because they are major HGF producers in the fetal liver. HGF is acrucial growth factor for hepatic development, and the factor isresponsible for liver parenchymal cell growth during liver regenerationas well. In addition, it has been shown that HpStCs, but not parenchymalcells, endothelial cells, and Kupffer cells, express HGF in adult liver.Therefore, our data and previous studies suggest that HpStCs are mainHGF producers from fetuses to adults in the liver. Thus, HpStCprecursors likely play a crucial role for hepatic and hematopoieticdevelopment in the fetal liver because the cells are main producers forHGF and SDF-1α.

Considering the unique phenotypic and functional characteristics ofHpStC precursors including expression of VCAM-1 and Hlx and productionof HGF and SDF-1α, the precursors might consist of a stem cell niche forhematopoietic stem cells or hepatic stem cells, or both in the liver.Because the serum-free culture system maintained the uniquecharacteristic phenotypes of HpStC precursors in vitro, the culturesystem can be use to develop an in vitro colony assay system to identifyHpStC precursors from adult livers. In addition, if a HpStCtransplantation system is developed, cell therapy using HpStC precursorswill be feasible. Identification, ex vivo expansion, and transplantationof HpStC precursors or HpStC progenitors in adult liver, would be avaluable resource to replace activated HpStCs in fibrogenic liver.Clearly, phenotypic identification and an in vitro culture system forHpStC precursors described in this study demonstrate a new direction todevelop novel therapeutic approaches for liver diseases.

The following examples are illustrative of the invention, but theinvention is by no means limited to these specific examples. A person ofordinary skill in the art will find in these examples but one means toimplement the instant invention. Further, while the instant exampleshave been presented in the context of rats for experimental convenience,the methods and reagents described herein can be readily translated tohuman application(s) by one of ordinary skill in the art from theteachings disclosed below.

Materials and Methods

Rats

Pregnant Fisher 344 rats were obtained from the Charles River BreedingLaboratory (Wilmington, Mass.). The morning on which the plug wasobserved was designated day 0. Male Fisher 344 rats (200-250 g) wereused for isolation of adult HpStCs. All animal experiments wereconducted under the institutional guidelines, and The University ofNorth Carolina Institutional Animal Care and Use Committee approved allexperimental procedures in accordance with The Guide for Care and Use ofLaboratory Animals of the National Academy of Sciences.

Cell Preparation

Hepatic progenitors suitable for in vitro propagation in accordance withthe instant invention are not limited to those isolated or identified byany particular method. In general, HpStC precursors may be obtained fromany excised section of liver. The excised section of liver may then bedissociated by standard procedures into single dissociated cells. Suchprocedures include enzymatic dissociation and/or mechanicaldissociation. Enzymatic dissociation may be carried out in the presenceof protease(s), such as collagenase(s), and/or nuclease(s), such asDNase. In some instances, pronase(s) may also be used. Methods ofenzymatic dissociation of liver cells are described and practiced in theart. By way of example, methods for the isolation and identification ofthe hepatic progenitors have been described in, for example, U.S. Pat.No. 6,069,005 and U.S. patent application Ser. Nos. 09/487,318;10/135,700; and 10/387,547, the disclosures of which are incorporatedherein in their entirety by reference. Indeed, various procedures existfor digestion and isolation of single cell suspensions of liver cells.It is to be understood, therefore, that the scope of the presentinvention is not to be limited to a specific method of procuring wholelivers or preparing single cell suspensions thereof.

In the instant Examples, fetal livers were isolated from 13˜14 dpc ratsand digested with 800 U/ml collagenase (Sigma) followed by furtherdigestion with Trypsin-EDTA solution (Sigma). The cell suspension wastreated with 200 U/ml DNase I (Sigma) (Kubota and Reid, 2000).

Cell Culture

In a preferred embodiment, the in vitro propagation steps involve usinga serum-free, hormone-supplemented, defined medium (HDM) to support thepropagation of HpStC precursor cells on a layer of feeder cells. Thefunction of the feeder cells is multi-fold, including supplyingnutrients, supplying an attachment surface, and secreting into themedium certain growth factors and extracellular matrix components neededfor survival, growth and/or differentiation of the precursor HpStCs. Thefeeder cells may be from reptiles, birds, crustaceans, fish, annelids,molluscs, nematodes, insects, or mammals, preferably human. Morepreferably, the feeder cells derive from embryonic tissue, and morepreferably, embryonic liver tissue. Fetal liver cells were cultured onSTO cell feeders and in a serum-free hormonally defined medium asdescribed previously (Kubota and Reid, 2000).

HDM consists of a 1:1 mixture of Dulbecco's modified Eagle's medium andHam's F12 (DMEM/F12, GIBCO/BRL) to which was added 2 mg/ml bovine serumalbumin (Sigma), 5 μg/ml insulin (Sigma), 10⁻⁶M dexamethasone (Sigma),10 μg/ml iron-saturated transferrin (Sigma), 4.4×10⁻³M nicotinamide(Sigma), 5×10⁻⁵M 2-mercaptoethanol (Sigma), 7.6 μeq/1 free fatty acid,2×10⁻³M glutamine (GIBCO/BRL), 1×10⁻⁶M CuSO₄, 3×10⁻⁸M Na₂SeO₃ andantibiotics (penicillin and streptomycin). Free fatty acids comprisedpalmitic, palmitoleic, stearic, oleic, linoleic, and linolenic acids(all Sigma) in the respective millimolar proportions of31.0:2.8:11.6:13.4:35.6:5.6 for 100 meq/l stock solution.

STO feeder cells were prepared as previously described (Kubota and Reid,2000). Briefly, a subclone of STO cells, STO5, was transfected withpEF-Hlx-MClneo. A transfected clone, STO5Hlx, were treated withmitomycin C (Sigma) and used for feeder cells at concentration of 2×10⁵cell per well in a 12-well plate. For long-term culture of sorted vA⁺cells, cells were cultured on STO feeders and in HDM supplemented with10 ng/ml human leukemia inhibitory factor (LIF; Boehringer Mannheim) and10 ng/ml epidermal growth factor (EGF; Collaborative BiomedicalProduct). Medium was changed every other day, and cells were subculturedto fresh STO feeders every week.

Immnocytochemical Staining of Colonies

Staining procedures for cultured cells were described previously (Kubotaand Reid, 2000). Briefly, culture plates were fixed in methanol-acetone(1:1) for 2 min at room temperature, rinsed and blocked with 20% goatserum (GIBCO/BRL) at 4° C. For double labeling of albumin (ALB) andcytokeratin (CK) 19, cultures were incubated with anti-rat ALB antibody(ICN Biomedicals) and anti-cytokeratin 19 (CK19) monoclonal antibody(Amersham) followed by Texas Red-conjugated anti-rabbit IgG (Vectorlaboratories) and FITC-conjugated anti-mouse IgG (Caltag). For nestin ordesmin expression, cells were stained with anti-nestin antibody(Rat-401, Developmental Studies Hybridoma Bank, The University of Iowa)or anti-desmin antibody (D33, Dako) followed by Alexa488-conjugatedanti-mouse IgG (Molecular Probes).

Fluorescence-Activated Cell Sorting (FACS)

Cells were analyzed and sorted by a FACStar Plus cell sorter (BDBiosciences) equipped with dual Coherent I-90 lasers. To detectvA-specific autofluorescence, cells were excited at 351 nm, andfluorescence emission was detected with the use of 450DF20 filter (OmegaOptical Inc, Brattleboro, Vt.). Fluorescence-conjugated antibodies wereexcited at 488 nm, and their fluorescence emission was detected bystandard filters.

Monoclonal antibodies used for analysis of rat cells wereFITC-conjugated anti-RT1A (B5; BD Biosciences), phycoerythrin(PE)-conjugated anti-rat ICAM-1 (1A29; BD Biosciences), anti-rat VCAM-1(5F10, Babco), anti-rat α6β1-integrin (mAB-5A, Serotec), anti-rat CD44(OX-49, BD Biosciences), PE-conjugated anti-rat VCAM-1 (MR109; BDBiosciences), PE-conjugated or biotin-conjugated anti-rat β3-integrin(2C9.G2; BD Biosciences), biotin-conjugated anti-rat PECAM-1 (TLD-3A12;BD Biosciences), biotin-conjugated anti-rat Thy-1 (OX-7; BDBiosciences). To block non-specific antibody binding, cells wereincubated with 20% goat serum (GIBCO/BRL), 1% teleostean gelatin(Sigma), and anti rat CD32 (FcγII receptor) antibody (D34-485, Rat BD FcBlock™, BD Biosciences) solution prior antibody staining in FACSexperiments. For staining with unconjugated anti-VCAM-1 antibody (5F10),fetal liver cells were incubated with the anti-VCAM-1 antibody followedby staining with biotin-conjugated anti-mouse IgG_(2a) monoclonalantibody (R19-15, BD Biosciences). Streptavidin-Cy-Chrome (BDBiosciences) was used to detect biotin-conjugated antibodies.

For the experiments of FACS to isolate long-term cultured cells thatwere derived from sorted vA⁺ cells, all cells in the culture wereharvested and stained with biotin conjugated anti-mouse CD98 (H202-141,BD Biosciences) followed by streptavidin-Cy-Chrome to separate culturedrat cells and STO feeder cells. Murine STO feeder cells were stainedbrightly with the antibody against mouse CD98. Thus, CD98-negative cellsrepresent rat-derived cells and could be distinguished readily usingFACS as shown previously (Kubota and Reid, 2000).

Colony Forming Assay (CFA) for Hepatoblasts

The procedure of CFA for hepatoblasts was described previously (Kubotaand Reid, 2000). Briefly, sorted cells were plated on STO feeders intriplicate at 500 or 2500 cells/well (3.8 cm²) in a 12-well plate andcultured in HDM for 14˜15 days with medium changes every other day. Toexamine bipotential differentiation activity of hepatoblasts, doubleimmunofluorescence staining of ALB and CK19 was performed. The colonieswere stained by Diff-Quick (Baxter) to count the number of the coloniesper well.

Cell Proliferation Assay

vA⁺ cells isolated by FACS were plated in triplicate at 500 cells/wellin a 96-well plates with HDM supplemented with laminin (CollaborativeBiomedical Products) at the final concentration of 8 μg/ml. EGF and LIFwere added at concentrations indicated. Five days after plating cellscultures were rinsed twice to remove floating cells and added freshmedium with the tetrazolium salt WST-1 (Boehringer Mannheim) to measurethe number of viable adherent cells (Kubota and Reid, 2000). After 4hours, the absorbance was determined according to the manufacturer'sprotocol.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

The primer sequences used for PCR are shown in Table 1.

TABLE 1 Number of Target Sequence amplification desminSense 5′-ATGAGCCAGGCCTACTCGTCC-3′ 35Anti-sense 5′-CAGCACTTCATGTTGTTGCTG-3′ nestinSense 5′-TGGAACAGAGATTGGAAGGCC-3′ 35Anti-sense 5′-CAGGAGTCTCAAGGGTATTAG-3′ vimentinSense 5′-TCCAACCGGAGCTATGTGACC-3′ 30Anti-sense 5′-CTCAGGTTCAGGGAAGAAAAG-3′ SMαASense 5′-ATGTGTGAAGAGGAAGACAGC-3′ 30Anti-sense 5′-GTGGTTTCGTGGATGCCCGC-3′ albuminSense 5′-ATGAAGTGGGTAACCTTTCTCC-3′ 26Anti-sense 5′-TGTGATGTGTTTAGGCTAAGGC-3′ Prox-1Sense 5′-GGGGAAAACCACAATTTCCACAC-3′ 33Anti-sense 5′-CCAGGAAGGATCAACATCTTTGC-3′ SDF-1αSense 5′-ATGGACGCCAAGGTCGTCGC-3′ 30Anti-sense 5′-GAAAGGGTCTCTGAGCACAG-3′ HGFSense 5′-TGGACAAGATTGTTATCGTGG-3′ 33Anti-sense 5′-ACGATTTGGGATGGCACATCC-3′ H1xSense 5′-CCTCGGTCCAGTCTATAAACC-3′ 30Anti-sense 5′-CAGCCGTTCTGAGGGCGAAGC-3′ β3-integrinSense 5′-GATGAAAAAATTGGCTGGAGG-3′ 33Anti-sense 5′-GCAGGTGGCATTGAAGGACAG-3′ GFAPSense 5′-CTCAATGACCGCTTTGCTAGC-3′ 35Anti-sense 5′-ACCACGATGTTCCTCTTGAGG-3′ β-actinSense 5′-ATGGATGACGATATCGCTGCG-3′ 26Anti-sense 5′-GGGTGTAAAACGCAGCTCAGTAA-3′

The procedure of RT-PCR for sorted cells by FACS was describedpreviously (Kubota et al., 2002). Briefly, cells were isolated using aFACStar Plus cell sorter, and total RNAs were extracted by RNeasy Kit(QIAGEN) and subjected to cDNA synthesis. cDNAs were synthesized fromtotal RNAs by oligo-dT priming and AMV reverse transcriptase (SeikagakuAmerica) in a reaction volume of 20 μl at 42° C. (Kubota et al., 2002).PCR was performed in a total volume of 50 μl consisting of 1 μM eachprimer, 200 μM each dNTP, 50 mM KCl, 1.5 mM MgCl₂, 10 mM Tris HCl, pH8.3, and 1.25 U Amplitaq polymerase gold (Perkins Elmer) withsynthesized cDNA. Samples were heated to 94° C. for 3 min followed byamplification for 26-35 cycles of 2 min at 94° C., 2 min 62° C., and 3min at 72° C. The number of amplification cycles for each target genewas varied and indicated in Table 1. After the last cycle, a finalextension step was done at 72° C. for 6 min. Then, 5 μl of each PCRreaction was analyzed by 1 agarose gel electrophoresis. cDNAssynthesized from total RNAs of sorted cells were normalized by the cellnumber.

Results

Identification of Vitamin A⁺ Cells in Fetal Liver

Once a single cell suspension has been established, isolation of HpStCprecursors involves exposing the mixed liver cell populations derivedfrom liver tissue to flow cytometry and selecting those cells thatexhibit specific auto-fluorescence generated by cytoplasmic vitamin A(vA) rich lipid droplets. Vitamin A specifically produces a green-bluefluorescence when excited with light of 330-360 nm (ultra violet, UV)laser. FACS analysis is able to detect the vitamin A-specific green-bluefluorescence (vA⁺) in the cytoplasm of both mature HpStCs as well asprecursor HpStCs in liver using a UV laser.

FIG. 1A shows the pattern of autofluorescence in the 13 dpc fetal livercells. The vA-specific, blue-green autofluorescent signal was measuredby detecting the emission light with a 450 nm filter by excitation of aUV laser (351 nm). To detect a non-UV laser-specific autofluorescentsignal, a 488 nm laser and 530/30 nm bandpass filter was used. Patternsof the autofluorescent signals of whole fetal liver cell population aswell as two subpopulations (R1 and R2 gates of FIG. 1A) are shown inFIG. 1B. In the FACS pattern of the whole cell population (FIG. 1B ALL),two distinct subpopulations with high autofluorescent characteristicswere identified. One had an autofluorescent signal specific for UV light(FIG. 1B ALL, upper left), which is referred to as vA⁺ here, whereascells locating diagonally in the upper right quadrant indicatenon-specific autofluorescence, because the autofluorescent signals weredetected with the 530 nm filter and the 450 nm filter when excited bythe 488 nm laser and the UV laser, respectively. The subpopulation withnon-specific autofluorescent characteristics (designated as ns-autoflu⁺)exclusively derived from the SSC^(high) gate (FIG. 1A, R2 and FIG. 1B,R2) while vA⁺ cells (FIG. 1B, upper left) were detected in both R1 andR2.

FIG. 1C shows the pattern of vA-specific autofluorescent signal and MHCclass Ia expression, which was detected by a FITC-conjugated antibodyagainst RT1A. FACS analysis indicated that vA⁺ cells as well asns-autoflu⁺ cells had no RT1A expression, because those two populationsdid not shift in the stained sample (FIG. 1C, R2) compared to thecontrol sample (FIG. 1B, R2). In addition, FACS analysis also indicatedthat the hepatoblast population, cells that are RT1A⁻ICAM-1⁺SSC^(high)(FIG. 1A R2, lower right) and RT1A⁻ns-autoflu⁺ cells (FIG. 1C R2, arrow)are an overlapping population by this FACS analysis.

To determine whether these autofluorescent signals were specific infetal liver, fetal lung cells from the 13 dpc fetuses were isolated andanalyzed by FACS. The FACS analysis showed there were neitherns-autoflu⁺ cells nor vA⁺ cells in the lung cells (FIG. 1D), indicatingthat the autofluorescent signals in particular subpopulations in thefetal liver were unique phenotypic characteristics.

As hepatic progenitor cells (i.e., hepatoblasts) have been suggested tobe RT1A⁻ OX18^(low)ICAM-1⁺SSC^(high) cells in 13 dpc liver of rat fetus,these markers were assayed in vA⁺ cells. FIG. 1A shows the patterns ofFACS analysis of fetal liver cells at 13 dpc followed staining withantibodies against RT1A, rat MHC class I, and ICAM-1. FIG. 1C shows thepattern of vA⁻ specific autofluorescent signal and RT1A expression,which was detected by FITC-conjugated antibody against RT1A. FACSanalysis indicated that vA⁺ cells as well as ns-autoflu⁺ cells had noRT1A expression, because those two populations did not shift in thestained sample (FIG. 1C, R2) compared to the control sample (FIG. 1B,R2). In addition, FACS analysis indicated that the hepatoblastpopulation, cells that are RT1A⁻ICAM-1⁺SSC^(high), (FIG. 1A, R2, lowerright) and RT1A⁻ns-autoflu⁺ cells (FIG. 1C, R2, arrow) were an identicalpopulation. These results indicate that FACS analysis was able to detectcharacteristic vA⁺ cells in rat fetal liver as early as 13 dpc and thatthe vA⁺ cells were RT1A-ICAM-1⁻.

Vitamin A⁺ Cells Express VCAM-1 and Integrin β3

As demonstrated above, vA positivity and MHC class Ia negativity aresufficient markers to identify and isolate HpStCs. However, in somecircumstances UV selection based on UV light may not be desirable,particularly where molecular (e.g., DNA) integrity is of concern.Therefore, the present invention provides markers that may be used inaddition to or in lieu of UV-based selection. HpStC precursors can befurther identified by exposing the selected cell population toantibodies specific for VCAM, more specifically VCAM-1. VCAM-1 issignificant because it has been shown to be a unique surface markerdistinguishing HpStCs from myofibroblasts in adult liver. As well, theexpression of VCAM-1 appears to be developmentally controlled becauseadult hepatocytes are negative for this marker.

VCAM-1 expression was analyzed in fetal liver cells to investigatewhether the vA⁺ cells express VCAM-1. By FACS analysis, it appeared thatabout 15% of cells were VCAM-1⁺ in the 13 dpc fetal liver (FIG. 2A). Thepattern of autofluorescence and RT1A expression of the VCAM-1⁺ cells wasnext determined. VCAM-1⁺ cells contained essentially all vA⁺ cells aswell as the entire ns-autoflu+ cell population (FIG. 2A), indicatingthat HpStCs and hepatoblasts express VCAM-1. FACS analyses of twomonoclonal antibodies against rat VCAM-1 (5F10 and MR109) showed anidentical pattern of VCAM-1 expression. In addition, fetal liver VCAM-1⁺cells were RT1A⁻ICAM-1⁺ cells because the R1 gate in FIG. 2B includedthe VCAM-1⁺ cell population. These results suggest that fetal liverVCAM-1⁺RT1A⁻ICAM-1⁺ cells consist of vA⁺ cells, hepatoblasts, and somenon-autofluorescent cells.

Additional surface antigens were investigated to distinguish the twoautofluorescent populations, the vA⁺ cells and the hepatoblasts, both ofwhich were VCAM-1⁺RT1A⁻ICAM-1⁺ cells. Because β3-integrin (CD61) isexpressed on endothelial cells, vascular smooth muscle cells, and adultHpStCs, two-color FACS analyses of VCAM-1 versus integrin (33 wereperformed. The majority of vA⁺RT1A⁻ cells expressed β3-integrin whereasns-autoflu⁺RT1A⁻ cells were β3-integrin-, while vA⁻RT1A⁻ cells containedsome VCAM-1⁺β3-integrin⁻ cells (FIG. 3B).

Autoflu⁻RT1A⁻ cells contained some VCAM-1⁺ β3-integrin⁺ cells. Theremaining major population (FIG. 3B, R4) was VCAM-1⁻ and appeared to becorrespond to R2 cell population in FIG. 2B. The R4 cell populationcomprised non-adherent cells when they were cultured on plastic dishes,suggesting that they were hematopoietic cells. A subpopulation (˜20%) ofthe fraction was β3-integrin⁺. Expression of PECAM-1 (CD31), which isknown as an endothelial cell marker, was also assessed. However, FACSanalysis indicated that PECAM-1 expression in vA⁺RT1A⁻ cells andns-autoflu⁺RT1A⁻ cells was negligible (FIG. 3B), while PECAM-1⁺ cellswere detected in the autoflu⁻RT1A⁻ and non-adherent cell populations(FIG. 3B, R2 and R4). Expression of Thy-1 (CD90), a surface marker foroval cells that appear in adult livers after oncogenic insults, wasfurther assessed. FACS analysis showed that ns-autoflu⁺ RT1A⁻ areThy-1⁻.

By contrast, vA⁺ RT1A⁻ cells, autoflu⁻RT1A⁻ cells and non-adherent cellsexpress Thy-1 heterogeneously. FACS analysis indicated that ns-autoflu⁺RT1A⁻ cells were CD44^(lo) whereas vA⁺ RT1A⁻ cells were CD44⁻ (data notshown). Although CD44 (Pgp-1) appeared to be expressed differentially inthe vA⁺ RT1A⁻ cells and ns-autoflu⁺ RT1A⁻, the expression on the cellsurface was weak. Together, these data suggest that β3-integrin antibodystaining, among all antibodies examined, facilitate distinguishing thevA⁺ RT1A⁻ cells and ns-autoflu⁺ RT1A⁻ cells, both of which populationswere VCAM-1⁺ ICAM-1⁺ in the fetal livers.

VCAM-1⁺ Integrin β3⁻ Non-Specific Autofluorescent Cell PopulationContains Only Hepatoblasts

Fetal hepatic cells of the rat until 14 dpc are homogeneous withdevelopmental potential to differentiate to both the hepatocytes andbiliary epithelial cells depending upon the microenvironment. Thesebipotent progenitors are called hepatoblasts. To examine whether vA⁺cells have any potential to generate hepatic cell lineages, the CFA(Kubota and Reid, 2000) was performed. Four cell populations wereisolated by FACS and subjected to the CFA for hepatoblasts: 1)ns-autoflu⁺RT1A⁻VCAM-1⁺ β3-integrin⁻ 2) vA⁺RT1A⁻VCAM-1⁺ 3) autoflu⁻RT1A⁻and 4) VCAM-1⁻ non-adherent cells. Sorted cell fractions were placed onSTO5 feeders in HDM, cultured for 15 days, and stained with antibodiesagainst albumin and CK19 for hepatic and biliary lineages, respectively.Then, all hepatic colonies were counted.

The CFA indicated that hepatic colonies were generated from group 1,ns-autoflu⁺ VCAM-1⁺ β3-integrin⁻ cells (Table 2, below), demonstratingthat the other groups of sorted cells, including the vA⁺VCAM-1⁼β3-integrin⁺ cells, are not hepatic progenitors. More than 95% of thehepatic colonies derived from the group 1-sorted cells contained bothhepatocytic (albumin⁺ CK19⁻) and biliary epithelial (albumin⁻CK19⁺)cells (FIG. 4). Further, the colony forming efficiency in the sortedns-autoflu⁺VCAM-1⁻ β3-integrin⁻cells was approximately 31%, and ahepatic progenitor cell line (rhe14321) established in a previous study(Kubota and Reid, 2000) had a colony efficiency in the CFA of 42.5±1.8%.Taken together, the result of CFA in this experiment indicated that thethe ns-autoflu⁺VCAM-1⁺β3-integrin⁻ cells population is a nearly purehepatoblast population, because CFAs by established cell lines ispresumably much higher than that of freshly isolated cells.

Table 2 provides the frequency of hepatic stellate colonies from sortedrodent fetal liver cells. Gates for fractionation of vA⁺RT1A⁻VCAM-1⁺,autoflu⁻RT1A⁻, ns-autoflu⁺RT1A⁻ and VCAM-1⁻ cells were created as shownin FIG. 3 R1, R2, R3 and R4, respectively.

Inoculated Hepatic Colony cell colony efficiency Cell Population numbernumber (%) vA⁺RT1A⁻VCAM-1⁺β3- 2500 (6) 3.3 ± 0.9 0.1 ± 0.0 integrin⁺ ¶autoflu⁻RT1A⁻ 2500 (6) 5.5 ± 0.2 0.2 ± 0.1 ns-autoflu⁺RT1A⁻VCAM-1⁺β3- 250 (6) 77.0 ± 6.7  30.8 ± 2.7  integrin⁻ § VCAM-1⁻ † 2500 (3) 0.0 ±0.0 0.0 ± 0.0 ¶: VCAM-1⁺β3-integrin⁺ cells from the R2 were sorted. §:VCAM-1⁺β3-integrin⁻ cells from the R1 were sorted. †: VCAM-1⁻ cells fromthe R4 were sorted.Flow cytometrically sorted cells were cultured on STO feeders atindicated cell numbers per well in a 12-well plate. The hepatic colonynumber is the average per well. Colony efficiency is expressed as thepercentage of cells inoculated in culture and that went on to formcolonies after 15 days of culture. Values are mean±SEM. Number of totalwell inoculated sorted cells is enclosed in parentheses.Gene Expression of Freshly Isolated Vitamin A⁺ VCAM-1⁺ Integrin β3⁺Cells

Gene expression pattern of the vA⁺ RT1A⁻VCAM-1⁺ β3-integrin⁺ cells wasnext assayed to examine whether they express various markers for HpStCs.Five population were isolated by FACS, and RNAs were isolated from thefive populations. RT-PCR for HpStC markers was perform using cDNAssynthesized from the RNAs. The five populations were: 1) ns-autoflu⁺RT1A⁻VCAM-1⁺ β3-integrin⁻, 2) vA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺, 3)autoflu⁻RT1A⁻VCAM-1⁺, 4) autoflu⁻RT1A⁻VCAM-1⁻, and 5) VCAM-1⁻non-adherent cell population. HpStCs in adult liver express intermediatefilaments, desmin and nestin, which are not expressed in other celltypes in the liver.

RT-PCR analyses showed that vA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺,ns-autoflu⁺RT1A⁻VCAM-1⁺, and autoflu⁻VCAM-1⁻ cells expressed all fourintermediate filaments. ns-autoflu⁺RT1A⁻VCAM-1⁺ cells express albumin aswell as Prox1, which is a transcriptional factor expressing specificallyin hepatoblasts. This result was consistent with the data obtained fromthe CFA assays, which demonstrated this population comprisedhepatoblasts. There was no expression of nestin, SMαA, or vimentin inthe hepatoblast population. The expression of HpStC specificintermediate filaments strongly suggests that vA⁺ cells are HpStCprecursors.

Subsequently, expression of three separate mesenchymal cell markers,HGF, stromal cell-derived factor-1 alpha (SDF-1α), and divergenthomeobox transcriptional factor, Hlx, were investigated using RT-PCR.HGF is required for normal hepatic development, especially forproliferation and differentiation of hepatoblasts in the mouse, and inadult liver HpStCs are major producers of HGF. SDF-1α is a potentchemokine for hematopoietic progenitors, and hematopoietic stem cells infetal liver migrate in response to the chemokine. Hlx is expressed inmesenchymal cells in developing fetal liver and plays an indispensablerole in fetal liver hematopoiesis and hepatic development.

Interestingly, vA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺ cells expressed HGF, SDF-1αand Hlx transcripts most strongly among all cell fractions examined(FIG. 5). Collectively, the vA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺ cells aredesmin⁺ nestin⁺ SMαA⁺ vimentin⁺ Hlx⁺ and are main producers for HGF andSDF-1α in fetal liver.

Ex Vivo Clonal Expansion of RT1A⁻VCAM-1^(+β) 3-Integrin⁺ vA⁺ Cells

HpStCs isolated from adult liver have only limited proliferativeactivity in vitro. The ex vivo growth capability of the vA⁺RT1A⁻VCAM-1⁺β3-integrin⁺ cells in fetal livers was investigated, because HpStCprecursors may have extensive proliferative activity. LIF is apleiotrophic growth factor for many different types of cells includingembryonic stem cells or myogenic cells. When vA⁺RT1A⁻VCAM-1⁺β3-integrin⁺ cells, which were isolated by FACS, were cultured with ahormonally defined serum-free medium at a cell density of 500 cells/wellof 96 well-plates for 5 days in the presence of LIF, the cells expandedin a dose-dependent manner (FIG. 6A). In addition, EGF, a growth factorfor various cell types including neural stem cells, enhanced theproliferation of HpStC precursors induced by LIF, but did not supportthe expansion on its own (FIG. 6A).

The proliferation, however, did not persist in the condition usingplastic culture plates alone. Therefore, the FACS-sorted vA⁺RT1A⁻VCAM-1⁺β3-integrin⁺ cells were next placed on STO5 feeders (Kubota and Reid,2000). Although LIF is produced by STO cells, exogenous LIF andsupplementation of EGF further supported colony formation from sortedvA⁺RT1A⁻VCAM-1⁺ β3-integrin⁺ cells dramatically (FIG. 6B). Proliferatingcells in the culture expressed desmin and nestin, whereas STO5 feedersdid not express either (FIG. 7). Three single colonies were picked andplaced on fresh STO5 feeders. The single colony-derived cells continuedto proliferate in the co-cultures with STO5 feeders supplemented withLIF and EGF for 2 months, indicating that they have extensive growthpotential. Expression of desmin and nestin were maintained in theproliferating cells (FIG. 8).

To compare further the characteristic phenotypes of 2 month-culturedcells with freshly isolated vA⁺ RT1A⁻VCAM-1⁺ β3-integrin⁺ cells, RT-PCRwas performed. Single colony-derived cells (A428-3) that were maintainedfor 2 months in culture were separated from STO5 feeder cells by FACS,and the RNA was extracted for RT-PCR analysis. RNA was isolated fromSTO5 feeder cells that were sorted simultaneously for a control sample.In addition, RNA was isolated from adult HpStCs to compare with thosefrom A428-3 and STO5 cells.

The results demonstrated that A428-3 expressed desmin, nestin, SMαA,vimentin, β3-integrin, SDF-1α, HGF, and Hlx, indicating the expressionpattern was a similar to fresh vA⁺ RT1A⁻VCAM-1⁺ β3-integrin⁺ cells (FIG.5 and FIG. 9A). Furthermore, VCAM-1 expression was confirmed by FACSanalysis (FIG. 9B). RT1A expression appeared to be induced in vitroculture (FIG. 9B). The RT-PCR results of adult HpStCs agreed withprevious reports, in which the phenotype of normal adult HpStCs isdesmin⁺, glial fibrillary acidic protein (GFAP)⁺, HGF⁻, but SMaA^(lo/−).The results also showed that adult HpStCs express SDF-1α, β3-integrin,and Hlx. There was neither expression of GFAP in A428-3 cells (FIG. 9A)or in any fractions tested in fetal liver.

The present invention, however, provides additional markers that may beused in conjunction with the aforementioned markers to identify HpStCprecursor cells, including, for example, β3-integrin+PECAM-1-VLA-6+ andCD44H−. ICAM-1 and β3-integrin are expressed on mature HpStCs as well.In addition to those surface markers, both mature and precursor HpStCsexpress intermediate filaments specific for HpStCs including desmin,vimentin, smooth muscle a-actin and nestin.

In this study, there was no detectable expression of GFAP in A428-3cells (FIG. 9A) or in any fractions tested in fetal liver GFAP. AlthoughGFAP is a marker used to identify astrocytes in central nervous system,the protein is also expressed in HpStCs in adult liver. However, we didnot find GFAP mRNA by RT-PCR in any cell fractions examined as well asthe whole fetal liver sample. In addition, even after culture ofisolated HpStC precursors, GFAP expression was not induced, whereasdesmin and nestin expression was sustained in the culture. This resultsuggests that HpStC precursors in fetal liver will acquire GFAPexpression in a later developmental stage. We cannot, however, excludeanother possibility, in which GFAP+ cells are derived from differentprecursors that do not exist in the 13 dpc fetal liver. Circulatingcells in the blood flow may be a source of the alternative cellularorigin. However, the majority of HpStCs in adult liver express GFAP;therefore, the minor contribution of circulating cells from the bloodare unlikely to become a dominant population in the liver. Thus, itseems more likely that acquisition of GFAP expression happens duringmaturation of HpStCs.

The data also indicated that HpStC precursors expressed the divergenthomeobox protein, Hlx, relatively strongly. Although the relationshipbetween Hlx expression and HpStC development is not clear, loss of Hlxexpression may contribute to the defects in the mutant mice. HpStCprecursors in the mouse fetal liver express HGF, SDF-1α and Hlx as well.

In addition to the unique surface phenotype of HpStC precursors, theculture system established in this study can be use to identify HpStCprecursors in adult liver. Until now, HpStCs from adult liver have beencultured in medium supplemented with fetal bovine serum. Normally,HpStCs cultured in the serum-supplemented medium give rise tomyofibroblastic cells, which acquired fibroblastic characteristics andlose the original HpStC phenotypes. Therefore, the serum-supplementedmedium conditions are not appropriate to identify HpStC precursors. Theserum-free culture conditions described in this study support ex vivomaintenance of HpStC progenitors.

It seems that HpStC precursors plays key roles for liver development,because they express more HGF transcript than any subpopulation in fetalliver cell fractions examined. HGF is a crucial growth factor forhepatic development (Schmidt et al., 1995), and the factor isresponsible for liver parenchymal cell growth during liver regenerationas well (Michalopoulos and DeFrances, 1997). In addition, it has beenshown that HpStCs, but not parenchymal cells, endothelial cells, orKupffer cells, are the producer for HGF in adult liver (Schirmacher etal., 1992). Therefore, our data and that from previous studies suggestthat HpStCs are the main HGF producers from fetuses to adults in theliver.

In this study, there was no GFAP expression in 13 dpc fetal liver.Although GFAP is a marker used to identify astrocytes in central nervoussystem, the protein is also expressed in HpStCs in adult liver. However,we did not find GFAP mRNA by RT-PCR in any cell fractions examined aswell as the whole fetal liver sample. In addition, even after culture ofisolated HpStC precursors, GFAP expression was not induced, whereasdesmin and nestin expression was sustained in the culture. This resultsuggests that HpStC precursors in fetal liver will acquire GFAPexpression in a later developmental stage. We cannot, however, excludeanother possibility, in which GFAP⁺ cells are derived from differentprecursors that do not exist in the 13 dpc fetal liver. Circulatingcells in the blood flow may be a source of the alternative cellularorigin. However, the majority of HpStCs in adult liver express GFAP;therefore, the minor contribution of circulating cells from the bloodare unlikely to become a dominant population in the liver. Thus, itseems more likely that acquisition of GFAP expression happens duringmaturation of HpStCs.

A divergent homeobox protein, Hlx, is expressed in the septumtransversum and mesenchymal cells in fetal liver (Lints et al., 1996). Aprevious study of Hlx knockout mice demonstrated that the mutant micehave impaired hepatic development and fetal liver hematopoiesis (Hentschet al., 1996).

Transplantation experiments indicated that the hematopoietic defect wascaused by the fetal liver microenvironment, but not by the hematopoieticprogenitors per se. Thus, Hlx⁺ cells are a crucial cell population infetal liver for supporting hepatic and hematopoietic development. Ourdata indicated that HpStC precursors expressed Hlx strongly. Therefore,it is interesting to examine whether Hlx knockout mice have HpStCprecursors. Although the relationship between Hlx expression and HpStCdevelopment is not clear, loss of Hlx expression may contribute thedefects in the mutant mice. Recently, we found that similar HpStCprecursors in the mouse fetal liver expressed HGF, SDF-1a, and Hlx aswell. Further, the present inventors have identified mesenchymal cellswith similar markers (e.g., smooth muscle alpha-actin) in human fetallivers and that have proven vital for the ex vivo expansion of humanhepatic stem cells.

HpStC precursors that were purified by FACS proliferated on STO feedersand under serum-free media conditions supplemented with lipids, insulin,transferrin, EGF and LIF. Our data indicated LIF is more beneficial forin vitro proliferation. With the support of STO feeders, HpStCprecursors replicated continuously for more than 2 months. Culturedcells expressed VCAM-1, β-3-integrin, desmin, vimentin, smooth musclealpha-actin, nestin, HGF and SDF-1α. These phenotypes of fresh HpStCprecursors did not change during in vitro culture.

In addition to the unique surface phenotype of HpStC precursors, theculture system established in this study can be use to identify HpStCprecursors in adult liver. Until now, HpStCs from adult liver have beencultured in medium supplemented with fetal bovine serum. Normally,HpStCs cultured in the serum-supplemented medium give rise tomyofibroblastic cells, which acquired fibroblastic characteristics andlose the original HpStC phenotypes. Therefore, the serum-supplementedmedium conditions are not appropriate to identify HpStC precursors. Theserum-free culture conditions described in this study support ex vivomaintenance of HpStC progenitors. If there exist HpStC precursors inadult liver, they would be a valuable resource to replace activatedHpStCs in fibrogenic liver. Phenotypic identification and an in vitroculture system for HpStC precursors will facilitate the development ofnovel therapeutic approaches for liver diseases.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or alterations of the invention following. In general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

What is claimed is:
 1. A medium for propagating hepatic stellate cellsand precursors thereof comprising: (a) a 1:1 mixture of Dulbecco'smodified Eagle's medium and Ham's F12; (b) 2 mg/ml bovine serum albumin;(c) 5 μg/ml insulin; (d) 10⁻⁶ M dexamethasone; (e) 10 μg/mliron-saturated transferrin; (f) 4.4×10⁻³ M nicotinamide; (g) 5×10⁻⁵ M2-mercaptoethanol; (h) 7.6 μeq/l free fatty acid selected from the groupconsisting of palmitic, palmitoleic, stearic, oleic, linoleic, andlinolenic acid; (i) 2×10⁻³ M glutamine; (j) 1×10⁻⁶ M CuSO₄; (k) 3×10⁻⁸ MNa₂SeO₃; (l) penicillin and streptomycin; (m) 10 ng/ml human leukemiainhibitory factor (LIF); and (n) 10 ng/ml epidermal growth factor (EGF).2. A culture system for propagating hepatic stellate cells andprecursors thereof comprising: (a) the medium of claim 1; and (b) alayer of feeder cells.
 3. The culture system of claim 2, in which thefeeder cells are STO cells.